The present description refers to methods and kits for the assessment of RNA quality in a sample. Stable RNA is used as a reference for the assessment of RNA quality, wherein the stable RNA has low susceptibility to nuclease degradation.
Nucleic acid analysis is most important in molecular diagnostics and molecular research. For quantitative analysis the most popular and accurate technology is quantitative real-time polymerase chain reaction (qPCR). Specific DNA targets are easy to quantify with qPCR. The DNA molecule is stable, resistant, and easy to purify. RNA targets are much more challenging. The RNA molecule is not particularly stable. It is self-cleaved at low as well as high pH and at elevated temperature. It is susceptible to UV-light and many chemicals, which either cleave it or modify it such that the RNA cannot be reverse transcribed into cDNA for subsequent qPCR analysis, it is also prone to cleavage by nucleases. Degraded RNA may escape detection and partially degraded RNA may be erroneously quantified. Even in relative quantification, when the indicator is the relative amount of two RNAs, RNA integrity is essential, since degradation may affect the compared RNAs differently. The importance of RNA quality in quantitative analysis has led to the development of methods to assess sample integrity. By far most common method is electrophoresis. The RNA contained in a matrix is separated based on length in an electric field and stained. Initially agarose gels were used, but have been replaced by more powerful systems such as capillary electrophoresis and microfluidics represented by the Bioanalyzer (Agilent), Experion™ System (Bio-Rad), QIAxel (Qiagen) and the ScreenTape (Agilent). These systems analyze the electrophoretic traces based on which the integrity is quantified as the RNA Integrity Number (RIN), RNA quality indicator (RQI), RNA Integrity Number Equivalent (RINe) and equivalent. The approach works well, but suffers from the fact that most of the RNA, typically >80%, is ribosomal RNA (rRNA) and the electropherogram is dominated by the 18S and 28S rRNA species (in mammalian systems). Hence, the approach assesses the quality of rRNA rather than of the targeted mRNA. Since rRNA molecules are very different from mRNA molecules, for example they have neither 5′-CAP nor A-tail and they are complexed with ribosomal proteins in the cell, they may experience different degradation. Hence, the quality measure obtained from electrophoretic analysis of RNA may not be relevant for expression analysis.
A method to assess the integrity of mRNA directly is known in the art (WO 2005/090609 A1; Swift et al., BioTechniques 28 (2000) 524-531). The method is based on directed reverse transcription of the RNA molecule to produce cDNA. The reverse transcription is initiated at the mRNA 3′-end by hybridizing an oligo(T) primer to the A-tail of mRNA or by hybridizing a gene specific primer to a particular mRNA target. The cDNA is then amplified using two PCR assays targeting sequences at different distances from the RT primer. One PCR assay amplifies a sequence closer to the RT primer and hence to the mRNA 3′-end and the other PCR assay amplifies a sequence further away from the RT primer. The two PCR assays may share reverse primer, if they extend differently, but not the forward primer. If the mRNA was intact the RT primer is expected to be extended all the way to the mRNA 5′-end and the two PCR assays shall produce the same amount of amplicon, which is readily measured if the PCR is performed as qPCR. However, if the mRNA was partially degraded the RT primer could not be extended beyond the break-point and the assay further away from the priming site (closer to the mRNA 5′.end) would yield less amplicon than the assay nearer to the RT primer (closer to the mRNA 3′-end). In the literature this approach to assess RNA quality has become known as the 3′/5′ assay (Nolan et al., Nat. Protoc. 1 (2006) 1559-1582). The 3′/5′ assay performs well on rather pure material that is not too degraded. Most field samples are of poorer quality. If a sample contains fragmented nucleic acids or oligonucleotides (even very short ones), these will act as primers in the reverse transcription, and the effect of the necessary directional priming will be obscured. Since all degraded samples contain fragments that may prime the reverse transcription the dynamic range of the 3′/5′ assay is inherently limited. Another limitation of the 3′/5′ assay is that it only detects partially fragmented RNA; it will not reflect total loss of mRNA due to degradation. This is serious drawback since nucleolytic degradation, which is of major concern in biological samples, often degrades an mRNA fully once degradation is initiated. Exonucleases bind either the 3′ or 5′ end of the mRNA and degrades its full length to very short fragments. These fragments go undetected by the 3′/5′ assay.
In this context, the technical problem underlying the present description was to provide an improved method which overcomes the above drawbacks and allows for a more accurate assessment of the quality of the RNA used as a starting material for analysis and in particular for RT-qPCR experiments.